The Preferential Reconstitution of Receptor Proteins into Model Lipid Domains Studied by Atomic Force Microscopy


Nahoko Kasai, Chandra S. Ramanujan*, Kamal Marwaha*,
John F. Ryan*, and Keiichi Torimitsu
Materials Science Laboratory, *University of Oxford

 Receptor proteins play a fundamental role in biological membranes. They bind to ligand molecules and transfer signals into the cells by means of electricity and by chemical modification of proteins. Biological membranes are principally made of lipid molecules. Recent studies have revealed that lipid membrane in the synaptic junctions of central nervous system (CNS) is heterogeneous in composition and consists of small regions known as rafts [1]. These raft-like domains are loci for insertion of receptor proteins and are believed to act as signalling platforms that organize and compartmentalize receptor proteins to modify the synaptic signalling.
 We report atomic force microscopy (AFM) measurements of the glutamate receptors (GluRs), the most common membrane receptor proteins found in the CNS. GluRs are implicated in learning and memory, and up-regulation of its numbers in the post-synaptic membrane possibly being a key component of this process. In this work we have investigated the dependence of protein reconstitution in model membranes on lipid composition.
 We reconstituted purified GluRs into a lipid mixture system consisting of a mixture that makes up approximately 60% of the synaptic membrane. As shown in Fig. 1, the mixture formed two distinct domains when immobilized on mica: a high domain (HD) at 7 nm height and a low domain (LD) at 5 nm. The lateral extent of HD is typically 〜100 nm, which has structural similarities with the raft-like domains in synaptic membranes, albeit with much simpler composition. Then we reconstituted GluRs and found that the receptors preferentially insert into the HD comparing to the LD as shown in Fig. 2 [2]. This result demonstrates that bilayer thickness is a significant factor in the membrane self-assembly process. This can be expected to reveal signalling mechanism in synapses.
 This research was supported in part by Bio-nanotechnology IRC in UK and by the Strategic International Cooperative Program, Japan Science and Technology Agency (JST).

[1] J. A. Allen et al., Nat. Rev. Neurosci. 8 (2007) 128.
[2] C. S. Ramanujan et al., 52nd Biophys. Soc. Meeting Abst. (2008) B363.

Fig. 1. (A) AFM image of raft-like domains in lipid membrane on mica (2.5 µm square) and (B) height profile.
Fig. 2. AFM image of lipid domain with reconstituted membrane proteins (1 µm square).

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